A detailed design of stable-isotope techniques, NMR spectroscopy and mass spectrometry, for studying drug-biochemical receptor interactions in vitro is proposed in this project. The main emphasis is on unraveling the bio-organic mechanism of actions of alkylating agents in terms of their reactions with RNA and DNA. The agents include methyl methanesulfonate, dimethyl sulfate, 1-methyl-3-nitro-1-nitrosoguanidine, 1-methyl-1-nitrosourea and their ethyl analogs, dimethylnitrosamine (activated by microsomal enzymes), alpha-acetoxydimethylnitrosamine, ethylene oxide, aziridine, 2-chloroethylamine, bis-(2-chloroethyl)amine, bis-(2-chloroethyl)methylamine, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 2-chloroethyl isocyanate, cyclohexyl isocyanate. The reactions of both natural abundance and 13C-and 2H-enriched alkylating agents with mononucleotides, dinucleotides, RNA and DNA will be carried out in aqueous solution and maintained at neutrality by a pH-Stat, and the products will be unequivocally characterized by UV, IR, NMR and mass spectral analysis. The conformational variations will be determined by 1H-1H, 13C-1H, 31P-1H and 13C-31P coupling analysis. The relative chemical kinetics will be investigated by NMR using stable-isotope enriched agents. The alkylated product distribution will be calculated from NMR integration curves, and mass spectrometry using either GC-MS method or internal reference method. The reactive sites, relative chemical kinetics, conformational variations, cytotoxicity of alkylated products and product distribution will be utilized to correlate the structure-activity relationship.